Cancer metastasis to bone is common to many advanced cancers including breast, prostate, and lung cancers, causing severe pain, life-threatening hypercalcemia, and nerve compression syndromes. Under homeostatic conditions, bone-resorbing osteoclasts are tightly regulated for healthy bone turnover. In skeletal metastasis, however, tumor cells secrete soluble factors that promote osteoclast activity, resulting in the release of growth factors that stimulate local tumor growth. This leads to a vicious cycle of osteoclast activation and tumor cell proliferation, causing increased tumor burden and bone lesions. Thrombospondin-1 (TSP1) is a secreted protein that is known primarily for its role in inhibiting angiogenesis. There is evidence that TSP1 regulates nitric oxie (NO) signaling via two of its ligands, CD36 and CD47. Tonic levels of NO are critical for cell survival. Under pathological conditions, NO signaling is upregulated through iNOS. Specific regulatory pathways of iNOS have not been identified. TSP1 regulation of NO represents a promising novel therapeutic target for the treatment of primary and metastatic cancer. We propose to evaluate TSP1-mediated NO signaling in bone remodeling, both in healthy and bone metastatic states. We found that TSP1 deficient macrophages showed elevated iNOS transcript levels and increased NO activity. TSP1 depleted osteoclasts had differentiation defects consistent with misregulated NO signaling. TSP1-/- mice had significantly increased bone volume and reduced bone turnover, a defect rescued upon administration of a pan-NOS inhibitor. We have published that CD47-/- mice also have increased bone mass with associated osteoclast defects. Importantly, we found that, contrary to their roles in anti-angiogenesis, bone metastatic cell lines C42b and B16F10 have increased TSP1 expression. We hypothesize that TSP1 ligation of CD47 regulates NO signaling via inhibition of iNOS in osteoclasts and that tumor-derived TSP1 contributes to increased tumor-associated osteolysis in bone metastasis. The TSP1/CD47/NO signaling axis in healthy and metastatic bone remodeling will be evaluated using mouse models and mimetics and inhibitors to modulate TSP1/CD47 interaction and NOS activity. Significance: The research proposed in this research training plan will further the understanding of bone remodeling and elucidate the role of TSP1 regulation of NO signaling. Importantly, we will characterize the TSP1/CD47/NO signaling in bone under pathological conditions and provide valuable insight into the off-target effects of cancer therapeutics in trialto modulate this signaling pathway.